EPR-Derived Structure of a Paramagnetic Intermediate Generated by Biotin Synthase BioB.

Biotin (vitamin B7) is an enzyme cofactor required by organisms from all branches of life but synthesized only in microbes and plants. In the final step of biotin biosynthesis, a radical S-adenosyl-l-methionine (SAM) enzyme, biotin synthase (BioB), converts the substrate dethiobiotin to biotin through the stepwise formation of two C-S bonds. Previous electron paramagnetic resonance (EPR) spectroscopic studies identified a semistable intermediate in the formation of the first C-S bond as 9-mercaptodethiobiotin linked to a paramagnetic [2Fe-2S] cluster through one of its bridging sulfides. Herein, we report orientation-selected pulse EPR spectroscopic results that reveal hyperfine interactions between the [2Fe-2S] cluster and a number of magnetic nuclei (e.g., 57Fe, 15N, 13C, and 2H) introduced in a site-specific manner via biosynthetic methods. Combining these results with quantum chemical modeling gives a structural model of the intermediate showing that C6, the target of the second hydrogen-atom abstraction, is now in close proximity to the nascent thioether sulfur and is ideally positioned for the second C-S bond forming event.

[FeFe]-hydrogenase maturation: insights into the role HydE plays in dithiomethylamine biosynthesis.

HydE and HydG are radical S-adenosyl-l-methionine enzymes required for the maturation of [FeFe]-hydrogenase (HydA) and produce the nonprotein organic ligands characteristic of its unique catalytic cluster. The catalytic cluster of HydA (the H-cluster) is a typical [4Fe-4S] cubane bridged to a 2Fe-subcluster that contains two carbon monoxides, three cyanides, and a bridging dithiomethylamine as ligands. While recent studies have shed light on the nature of diatomic ligand biosynthesis by HydG, little information exists on the function of HydE. Herein, we present biochemical, spectroscopic, bioinformatic, and molecular modeling data that together map the active site and provide significant insight into the role of HydE in H-cluster biosynthesis. Electron paramagnetic resonance and UV-visible spectroscopic studies demonstrate that reconstituted HydE binds two [4Fe-4S] clusters and copurifies with S-adenosyl-l-methionine. Incorporation of deuterium from D2O into 5'-deoxyadenosine, the cleavage product of S-adenosyl-l-methionine, coupled with molecular docking experiments suggests that the HydE substrate contains a thiol functional group. This information, along with HydE sequence similarity and genome context networks, has allowed us to redefine the presumed mechanism for HydE away from BioB-like sulfur insertion chemistry; these data collectively suggest that the source of the sulfur atoms in the dithiomethylamine bridge of the H-cluster is likely derived from HydE's thiol containing substrate.

Biotin synthase: insights into radical-mediated carbon-sulfur bond formation.

The enzyme cofactor and essential vitamin biotin is biosynthesized in bacteria, fungi, and plants through a pathway that culminates with the addition of a sulfur atom to generate the five-membered thiophane ring. The immediate precursor, dethiobiotin, has methylene and methyl groups at the C6 and C9 positions, respectively, and formation of a thioether bridging these carbon atoms requires cleavage of unactivated CH bonds. Biotin synthase is an S-adenosyl-l-methionine (SAM or AdoMet) radical enzyme that catalyzes reduction of the AdoMet sulfonium to produce 5'-deoxyadenosyl radicals, high-energy carbon radicals that can directly abstract hydrogen atoms from dethiobiotin. The available experimental and structural data suggest that a [2Fe-2S](2+) cluster bound deep within biotin synthase provides a sulfur atom that is added to dethiobiotin in a stepwise reaction, first at the C9 position to generate 9-mercaptodethiobiotin, and then at the C6 position to close the thiophane ring. The formation of sulfur-containing biomolecules through a radical reaction involving an iron-sulfur cluster is an unprecedented reaction in biochemistry; however, recent enzyme discoveries suggest that radical sulfur insertion reactions may be a distinct subgroup within the burgeoning Radical SAM superfamily. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.

9-Mercaptodethiobiotin is generated as a ligand to the [2Fe-2S]+ cluster during the reaction catalyzed by biotin synthase from Escherichia coli.

Biotin synthase catalyzes formation of the thiophane ring through stepwise substitution of a sulfur atom for hydrogen atoms at the C9 and C6 positions of dethiobiotin. Biotin synthase is a radical S-adenosylmethionine (SAM) enzyme that reductively cleaves S-adenosylmethionine, generating 5'-deoxyadenosyl radicals that initially abstract a hydrogen atom from the C9 position of dethiobiotin. We have proposed that the resulting dethiobiotinyl radical is quenched by the µ-sulfide of the nearby [2Fe-2S](2+) cluster, resulting in coupled formation of 9-mercaptodethiobiotin and a reduced [2Fe-2S](+) cluster. This reduced FeS cluster is observed by electron paramagnetic resonance spectroscopy as a mixture of two orthorhombic spin systems. In the present work, we use isotopically labeled 9-mercaptodethiobiotin and enzyme to probe the ligand environment of the [2Fe-2S](+) cluster in this reaction intermediate. Hyperfine sublevel correlation spectroscopy (HYSCORE) spectra exhibit strong cross-peaks demonstrating strong isotropic coupling of the nuclear spin with the paramagnetic center. The hyperfine coupling constants are consistent with a structural model for the reaction intermediate in which 9-mercaptodethiobiotin is covalently coordinated to the remnant [2Fe-2S](+) cluster.

A complex between biotin synthase and the iron-sulfur cluster assembly chaperone HscA that enhances in vivo cluster assembly.

Biotin synthase (BioB) is an iron-sulfur enzyme that catalyzes the last step in biotin biosynthesis, the insertion of sulfur between the C6 and C9 atoms of dethiobiotin to complete the thiophane ring of biotin. Recent in vitro experiments suggest that the sulfur is derived from a [2Fe-2S](2+) cluster within BioB, and that the remnants of this cluster dissociate from the enzyme following each turnover. For BioB to catalyze multiple rounds of biotin synthesis, the [2Fe-2S](2+) cluster in BioB must be reassembled, a process that could be conducted in vivo by the ISC or SUF iron-sulfur cluster assembly systems. The bacterial ISC system includes HscA, an Hsp70 class molecular chaperone, whose yeast homologue has been shown to play an important but nonessential role in assembly of mitochondrial FeS clusters in Saccharomyces cerevisiae. In this work, we show that in Escherichia coli, HscA significantly improves the efficiency of the in vivo assembly of the [2Fe-2S](2+) cluster on BioB under conditions of low to moderate iron. In vitro, we show that HscA binds with increased affinity to BioB missing one or both FeS clusters, with a maximum of two HscA molecules per BioB dimer. BioB binds to HscA in an ATP/ADP-independent manner, and a high-affinity complex is also formed with a truncated form of HscA that lacks the nucleotide binding domain. Further, the BioB-HscA complex binds the FeS cluster scaffold protein IscU in a noncompetitive manner, generating a complex that contains all three proteins. We propose that HscA plays a role in facilitating the transfer of FeS clusters from IscU into the appropriate target apoproteins such as biotin synthase, perhaps by enhancing or prolonging the requisite protein-protein interaction.

Biochemical and Structural Properties of a Thermostable Mercuric Ion Reductase from Metallosphaera sedula.

Mercuric ion reductase (MerA), a mercury detoxification enzyme, has been tuned by evolution to have high specificity for mercuric ions (Hg(2+)) and to catalyze their reduction to a more volatile, less toxic elemental form. Here, we present a biochemical and structural characterization of MerA from the thermophilic crenarchaeon Metallosphaera sedula. MerA from M. sedula is a thermostable enzyme, and remains active after extended incubation at 97°C. At 37°C, the NADPH oxidation-linked Hg(2+) reduction specific activity was found to be 1.9 µmol/min⋅mg, increasing to 3.1 µmol/min⋅mg at 70°C. M. sedula MerA crystals were obtained and the structure was solved to 1.6 Å, representing the first solved crystal structure of a thermophilic MerA. Comparison of both the crystal structure and amino acid sequence of MerA from M. sedula to mesophillic counterparts provides new insights into the structural determinants that underpin the thermal stability of the enzyme.